Respiration from activity of respiratory chain: redoxsensor green
Template:BreadcrumbsSecondaryProduction
| Single-cell respiration (RSG) |
|---|
| Approach: fluorescent redox dye incorporation |
| Context: incubation, lab |
| Spatial scale: point sample |
| Temporal scale: minutes |
| Units: attomol O2 cell-1 h-1 (or µmol O2 L-1 h-1 at community level) |
| Community captured: single cell |
| Co-measurements: cell fluorescence, cell abundance (flow cytometry), O2 calibration (Winkler) |
Method Overview
RedoxSensor Green (RSG) is a cell-permeant, fluorogenic dye whose reduction is coupled to electron transport system (ETS) activity inside cells. Upon entering the cell, RSG is reduced by ETS enzymes to a highly fluorescent product that is retained intracellularly, and the resulting per-cell fluorescence intensity is proportional to respiration rate. Individual cells can then be analysed by flow cytometry or epifluorescence microscopy, enabling single-cell or population-level resolution of respiratory activity[1].
Conversion from RSG fluorescence to absolute oxygen consumption requires calibration against a parallel Winkler or optode-based bulk respiration measurement from the same sample. The assay is performed in discrete mode on natural seawater samples with no pre-concentration step.
Scale of measurement
The method provides a single point measurement in space. Incubations are short (minutes to one hour), minimizing the time cells spend outside their natural environment. Single-cell resolution means that community-level heterogeneity in respiratory activity can be characterized.
Data generated
Per-cell RSG fluorescence intensity, calibrated against bulk O2 consumption, yields single-cell respiration rates in attomol O2 cell-1 h-1. Community-level rates (µmol O2 L-1 h-1) are obtained by multiplying single-cell rates by cell abundance. The distribution of per-cell rates across the population reveals the proportion of highly active vs. low-activity cells.
Units & currency
Units are amol O2 cell-1 h-1 at the single-cell level, or µmol O2 L-1 h-1 for community-level estimates. The currency is oxygen.
Sample size
Typical samples are < 1 L in volume.
Repositories & databases
Limitations
RSG fluorescence is assumed to be proportional to respiratory rate across the range of physiological states encountered in the environment. In practice, cell membrane permeability — which governs RSG uptake — varies with the physiological state of individual cells, potentially biasing estimates in cells with compromised membranes or altered membrane potential. It is not established whether all active cells in a natural community incorporate RSG equally. The calibration step (linking fluorescence to O2 consumption via Winkler) introduces uncertainty from the assumption that the calibration applies across the diversity of cell types and physiological states in the sample.
Example Applications & Protocols
Classic examples
- Kalyuzhnaya et al. (2008) Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington [1]
Recent applications
- Munson-McGee et al. (2022) Decoupling of respiration rates and abundance in marine prokaryoplankton [2]
Common calculations/conversions
- Community respiration (µmol O2 L-1 h-1) = mean RSG fluorescence per cell × cell abundance × calibration factor (from parallel Winkler measurement).
References
- ↑ 1.0 1.1 Kalyuzhnaya, M. G., Lidstrom, M. E., & Chistoserdova, L. (2008). Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington. The ISME Journal, 2(7), 696–706. https://doi.org/10.1038/ismej.2008.32
- ↑ Munson-McGee, J. H., Lindsay, M. R., Sintes, E., Brown, J. M., D'Angelo, T., Brown, J., Lubelczyk, L. C., Tomko, P., Emerson, D., Orcutt, B. N., Poulton, N. J., Herndl, G. J., & Stepanauskas, R. (2022). Decoupling of respiration rates and abundance in marine prokaryoplankton. Nature, 612(7941), 764–770. https://doi.org/10.1038/s41586-022-05505-3